Unmethylated CpG motifs occur much more frequently in bacterial DNA than in vertebrate DNA. These motifs activate host defense mechanisms and lead to innate and acquired immune responses. CpG motifs and their immunostimulatory effects are reviewed in Krieg, Ann. Rev. Immunol. 20:709-760 (2002).
An immunostimulatory plasmid containing a number of CpG motifs was previously developed and has been shown to be effective for eliciting immune responses when administered to avian and bovine species in an immunomodulator composition comprising the plasmid and a cationic liposome delivery vehicle. See U.S. Patent Application Publications Nos. 2012/0064151 A1 (avian species) and 2013/0295167 A1 (bovine species), the contents of both of which are hereby incorporated by reference in their entirety. This plasmid, pMB75.6, is 4242 bp in length and contains 288 CpG dinucleotides. A map of pMB75.6 is shown in FIG. 1 and the nucleotide sequence of pMB75.6 is provided as SEQ ID NO: 2. As described in U.S. Patent Application Publication No. 2012/0064151, the immunomodulator composition containing pMB75.6 elicited a non-antigen-specific immune response that protected chickens from infectious disease when administered in ovo. This non-antigen-specific immune response was further enhanced with administration of at least one biological agent, such as a vaccine. In addition, the immunomodulator composition containing the pMB75.6 plasmid was found to have an adjuvant effect and to elicit an increase in the efficacy of vaccines. Similarly, as described in U.S. Patent Application No. 2013/0295167, the immunomodulator composition containing pMB75.6 elicited a non-antigen-specific immune response in cattle that protected the cattle from infectious disease.
However, as shown in FIG. 1, the pMB75.6 plasmid contains a kanamycin-resistance gene (KanR). Antibiotic-based selection and production systems are becoming increasingly disfavored due to concerns about horizontal transfer of the antibiotic resistance gene to bacteria in the environment. Potential horizontal transfer of antibiotic resistance genes is particularly concerning for vectors that are directly administered to a subject (e.g., immunostimulatory plasmids such as pMB75.6 or vectors used for gene therapy or DNA vaccination). There is therefore a need in the art for immunostimulatory plasmids that are capable of eliciting an immune response in a subject while also lacking antibiotic resistance genes.